Selective Affimers Recognise the BCL-2 Family Proteins BCL-xL and MCL-1 through Noncanonical Structural Motifs*.

The BCL-2 family is a challenging group of proteins to target selectively due to sequence and structural homologies across the family. Selective ligands for the BCL-2 family regulators of apoptosis are useful as probes to understand cell biology and apoptotic signalling pathways, and as starting points for inhibitor design. We have used phage display to isolate Affimer reagents (non-antibody-binding proteins based on a conserved scaffold) to identify ligands for MCL-1, BCL-xL , BCL-2, BAK and BAX, then used multiple biophysical characterisation methods to probe the interactions. We established that purified Affimers elicit selective recognition of their target BCL-2 protein. For anti-apoptotic targets BCL-xL and MCL-1, competitive inhibition of their canonical protein-protein interactions is demonstrated. Co-crystal structures reveal an unprecedented mode of molecular recognition; where a BH3 helix is normally bound, flexible loops from the Affimer dock into the BH3 binding cleft. Moreover, the Affimers induce a change in the target proteins towards a desirable drug-bound-like conformation. These proof-of-concept studies indicate that Affimers could be used as alternative templates to inspire the design of selective BCL-2 family modulators and more generally other protein-protein interaction inhibitors.

Synthesis and evaluation of a UMI-77-based fluorescent probe for selective detecting Mcl-1 protein and imaging in living cancer cells.

Development of efficient fluorescent probes for detecting the overexpressed Mcl-1 protein in living cells is imperative for the diagnosis and treatment of cancers. In this paper, a new UMI-77 based fluorescent probe (DNSH), was synthesized and characterized. DNSH bound to the hydrophobic pockets of Mcl-1 protein tightly and the binding affinity was 20-fold higher than that of previous developed Mcl-1 probe. DNSH exhibited specific fluorescence response to Mcl-1 protein rather than other proteins. In the presence of Mcl-1 protein, fluorescence emission of DNSH can be switched on. Furthermore, fluorescence colocalization experiment demonstrated that DNSH can be successfully used for imaging mitochondrial Mcl-1 protein in human prostate cancer cells without a washing process. These results showed that DNSH may find useful applications in biological research such as tracking Mcl-1 protein in living biological specimens.

Targeting MCL-1 in hematologic malignancies: Rationale and progress.

Myeloid cell leukemia sequence 1 (MCL-1) is an antiapoptotic protein that plays a key role in promoting cell survival in multiple myeloma (MM), acute myeloid leukemia (AML), and non-Hodgkin lymphoma (NHL). Overexpression of MCL-1 is associated with treatment resistance and poor prognosis; thus, MCL-1 inhibitors are rational therapeutic options for malignancies depending on MCL-1. Several MCL-1 inhibitors have entered clinical trials, including AZD5991, S64315, AMG 176, and AMG 397. A key area of investigation is whether MCL-1 inhibitors will complement the activity of BCL-2 inhibitors, such as venetoclax, and synergistically enhance anti-tumor efficacy when given in combination with other anti-cancer drugs. Another important question is whether a safe therapeutic window can be found for this new class of inhibitors. In summary, inhibition of MCL-1 shows potential as a treatment for hematologic malignancies and clinical evaluation of MCL-1 inhibitors is currently underway.

Immunohistochemical Biomarkers of Survival in Patients With Adenocarcinoma of the Uterine Cervix Receiving Chemoradiotherapy.

BACKGROUND/AIM: To determine the prognostic effects of immunohistochemical biomarkers for predicting chemoradiotherapy (CRT)-based treatment outcomes in patients with adenocarcinoma of the uterine cervix. MATERIALS AND METHODS: This study included 42 patients receiving definitive CRT. According to the International Federation of Gynecology and Obstetrics staging system, 13, 21, and 8 patients were classified as having stage IB2, II, and III disease, respectively. Baseline immunohistochemical biomarkers, including those for hypoxia, cell proliferation, cell adhesion, immunogenicity, and evasion of apoptosis, were analyzed using tissue microarrays from biopsy specimens. RESULTS: Myeloid cell leukemia-1 (MCL1) overexpression and the presence of pelvic lymph node metastasis were two prognostic factors for inferior cancer-specific survival. A higher H-score for c-MYC proto-oncogene, bHLH transcription factor (c-MYC) was associated with lower pelvic relapse-free survival. CONCLUSION: For patients with adenocarcinoma of the uterine cervix requiring definitive CRT, treatment outcomes can be stratified by the immunohistochemical biomarkers MCL1 and c-MYC for cancer death and local failure, respectively.

Discovery of a Turn-On Fluorescent Probe for Myeloid Cell Leukemia-1 Protein.

The environment-sensitive probe 2 with turn-on switch for Mcl-1 protein was developed herein. After careful evaluation, this small molecule fluorescent probe revealed a selective binding affinity with Mcl-1 protein with a Ki value of 2.6 µM and can be well applied to imaging Mcl-1 protein or detecting the cellular distribution of Mcl-1 protein inhibitors. Compared with other imaging approaches, such as the immunofluorescence and fluorescent protein-based techniques, this fluorescent method is rapid, convenient, and affordable, especially since a washing procedure is no longer needed. This environment-sensitive "off-on" design strategy may present a case in point for developing small-molecule fluorescent probe of Bcl-2 family proteins.

Intersection of mitochondrial fission and fusion machinery with apoptotic pathways: Role of Mcl-1.

Mitochondria actively contribute to apoptotic cell death through mechanisms including the loss of integrity of the outer mitochondrial membrane, the release of intermembrane space proteins, such as cytochrome c, in the cytosol and the caspase cascade activation. This process is the result of careful cooperation not only among members of the Bcl-2 family but also dynamin-related proteins. These events are often accompanied by fission of the organelle, thus linking mitochondrial dynamics to apoptosis. Emerging evidences are suggesting a fine regulation of mitochondrial morphology by Bcl-2 family members and active participation of fission-fusion proteins in apoptosis. The debate whether in mitochondrial morphogenesis the role of Bcl-2 family members is functionally distinct from their role in apoptosis is still open and, above all, which morphological changes are associated with cell death sensitisation. This review will cover the findings on how the mitochondrial fission and fusion machinery may intersect apoptotic pathways focusing on recent advances on the key role played by Mcl-1.

Re-activation of mitochondrial apoptosis inhibits T-cell lymphoma survival and treatment resistance.

T lymphocyte non-Hodgkin's lymphoma (T-NHL) represents an aggressive and largely therapy-resistant subtype of lymphoid malignancies. As deregulated apoptosis is a frequent hallmark of lymphomagenesis, we analyzed gene expression profiles and protein levels of primary human T-NHL samples for various apoptotic regulators. We identified the apoptotic regulator MCL-1 as the only pro-survival BCL-2 family member to be highly expressed throughout all human T-NHL subtypes. Functional validation of pro-survival protein members of the BCL-2 family in two independent T-NHL mouse models identified that the partial loss of Mcl-1 significantly delayed T-NHL development in vivo. Moreover, the inducible reduction of MCL-1 protein levels in lymphoma-burdened mice severely impaired the continued survival of T-NHL cells, increased their susceptibility to chemotherapeutics and delayed lymphoma progression. Lymphoma viability remained unaffected by the genetic deletion or pharmacological inhibition of all alternative BCL-2 family members. Consistent with a therapeutic window for MCL-1 treatment within the context of the whole organism, we observed an only minimal toxicity after systemic heterozygous loss of Mcl-1 in vivo. We conclude that re-activation of mitochondrial apoptosis by blockade of MCL-1 represents a promising therapeutic strategy to treat T-cell lymphoma.

Blockade of BCL-2 proteins efficiently induces apoptosis in progenitor cells of high-risk myelodysplastic syndromes patients.

Deregulated apoptosis is an identifying feature of myelodysplastic syndromes (MDS). Whereas apoptosis is increased in the bone marrow (BM) of low-risk MDS patients, progression to high-risk MDS correlates with an acquired resistance to apoptosis and an aberrant expression of BCL-2 proteins. To overcome the acquired apoptotic resistance in high-risk MDS, we investigated the induction of apoptosis by inhibition of pro-survival BCL-2 proteins using the BCL-2/-XL/-W inhibitor ABT-737 or the BCL-2-selective inhibitor ABT-199. We characterized a cohort of 124 primary human BM samples from MDS/secondary acute myeloid leukemia (sAML) patients and 57 healthy, age-matched controls. Inhibition of anti-apoptotic BCL-2 proteins was specifically toxic for BM cells from high-risk MDS and sAML patients, whereas low-risk MDS or healthy controls remained unaffected. Notably, ABT-737 or ABT-199 treatment was capable of targeting the MDS stem/progenitor compartment in high-risk MDS/sAML samples as shown by the reduction in CD34(+) cells and the decreased colony-forming capacity. Elevated expression of MCL-1 conveyed resistance against both compounds. Protection by stromal cells only partially inhibited induction of apoptosis. Collectively, our data show that the apoptotic resistance observed in high-risk MDS/sAML cells can be overcome by the ABT-737 or ABT-199 treatment and implies that BH3 mimetics might delay disease progression in higher-risk MDS or sAML patients.

Clinical activity of alvocidib (flavopiridol) in acute myeloid leukemia.

There have been minimal therapeutic advancements in acute myeloid leukemia (AML) over the past 4 decades and outcomes remain unsatisfactory. Alvocidib (formerly flavopiridol) is a multi-serine threonine cyclin-dependent kinase inhibitor with demonstrable in vitro and clinical activity in AML when combined in a timed sequential chemotherapy regimen, FLAM (alvocidib followed by cytarabine continuous infusion and mitoxantrone). FLAM has been evaluated in sequential phase 1 and phase 2 studies in 149 and 256 relapsed/refractory and newly diagnosed non-favorable risk AML patients, respectively, with encouraging findings in both patient populations warranting further investigation. This review highlights the mechanism of action of alvocidib, pre-clinical studies of alvocidib in AML, and the clinical trials evaluating alvocidib alone and in combination with cytotoxic agents (FLAM) in AML.

ApoG2 inhibits the antiapoptotic protein, Mcl­1, and induces mitochondria­dependent apoptosis in human colorectal cancer cells.

Colorectal cancer (CRC) is a worldwide malignancy of high incidence and mortality. At present, there is a lack of effective drugs against CRC. The B­cell leukemia/lymphoma 2 (Bcl­2) protein family members are considered to be closely associated with tumorigenesis and the chemoresistance of CRC. As a novel gossypol derivative targeting antiapoptotic proteins of the Bcl­2 family, apogossypolone (ApoG2) exhibits antitumor properties in various cancer types, although its effects against CRC remain to be fully elucidated. In the present study, the cytotoxicity of ApoG2 in vitro on CRC cells was investigated, with the aim of elucidating the underlying mechanism. Using an MTT assay, ApoG2 was revealed to inhibit the growth of the HT29, SW480 and HCT116 CRC cell lines in a dose­ and a time­dependent manner. Hoechst staining revealed that ApoG2 induced CRC cell apoptosis, marked by morphological changes, including cell shrinkage and nuclear fragmentation. Flow cytometric analysis also detected a higher apoptotic ratio following treatment with ApoG2. The ratio was dependent upon the concentration of ApoG2, which the cells were exposed to, and the duration of the exposure. Western blot analysis and immunoprecipitation experiments revealed that ApoG2 treatment led to the downregulation of the protein expression of Mcl­1, and the interruption of the binding of Mcl­1 to the protein Bax. Furthermore, treatment with ApoG2 led to the release of cytochrome c into the cytoplasm and the activation of caspases 3 and 7. The present study revealed that ApoG2 inhibited the proliferation of the CRC cell lines through mitochondrial signaling pathway­dependent apoptosis, which may be associated with the disruption of the function of the Mcl­1 protein by ApoG2.

mTOR Complex 2 Stabilizes Mcl-1 Protein by Suppressing Its Glycogen Synthase Kinase 3-Dependent and SCF-FBXW7-Mediated Degradation.

mTOR complex 2 (mTORC2) regulates cell survival and growth through undefined mechanisms. Mcl-1, a Bcl-2 family protein, functions as an oncogenic protein. The connection between mTORC2 and Mcl-1 stability has not been established and was thus the focus of this study. Mcl-1 levels in cancer cells were decreased by mTOR kinase inhibitors (TORKinibs), which inhibit both mTORCs, by knocking down rictor and by knocking out rictor or Sin1 but not by silencing raptor. TORKinib treatment and rictor knockdown did not alter Mcl-1 mRNA levels but rather decreased its protein stability. Moreover, TORKinib-induced Mcl-1 reduction was rescued by proteasome inhibition. Consistently, TORKinib increased Mcl-1 ubiquitination. Hence, it is clear that inhibition of mTORC2 enhances Mcl-1 degradation, resulting in Mcl-1 reduction. Suppression of glycogen synthase kinase 3 (GSK3) or FBXW7 rescued Mcl-1 reduction induced by TORKinibs or rictor knockdown. Thus, mTORC2 inhibition apparently induces Mcl-1 degradation through a GSK3-dependent and SCF-FBXW7-mediated mechanism. Intriguingly, we detected a direct association between mTORC2 and SCF-FBXW7; this association could be inhibited by TORKinib treatment, suggesting that mTORC2 may directly associate with and inhibit the SCF-FBXW7 complex, resulting in delayed Mcl-1 degradation. Collectively, our findings highlight a novel mechanism by which mTORC2 regulates cell survival and growth by stabilizing Mcl-1.

Myeloid cell leukemia-1 regulates the cell growth and predicts prognosis in gastric cancer.

The expression of myeloid cell leukemia-1 (Mcl­1), a member of the anti-apoptotic Bcl-2 protein family, has been associated with tumor progression and adverse patient outcome. The aims of current study were to evaluate whether Mcl-1 affects the survival or death of gastric cancer cells, and to investigate the prognostic value of its expression in gastric cancer. PcDNA3.1-Mcl-1 expression and Mcl-1 siRNA vectors were used to overexpress and silence Mcl-1 expression in gastric cancer cell lines including SNU638 and TMK1, respectively. Immunohistochemistry was used to determine the expression of Mcl-1 in gastric cancer tissues. Apoptosis was determined by the TUNEL assay, and cell proliferation was determined by immunostaining with a Ki-67 antibody. Mcl-1 knockdown induced apoptosis through the upregulation of caspase-3, and -7, and PARP activity, and the release of Smac/DIABLO and Omi/HtrA2 into the cytoplasm. Additionally, cell cycle arrest occurred due to decrease of cyclin D1, cell division cycle gene 2 (cdc2), and cyclin-dependent kinase 4 and 6. In contrast, overexpression of Mcl-1 inhibited apoptosis and cell cycle arrest. Mcl-1 knockdown did not suppress tumor cell proliferation in gastric cancer cells, whereas overexpression of Mcl-1 enhanced tumor cell proliferation. The JAK2 and STAT3 signaling cascades were significantly blocked by Mcl-1 knockdown. The mean Ki-67 labeling index (KI) value of Mcl-1 positive tumors was significantly lower than that of Mcl-1 negative tumors. However, there was no significant difference between Mcl-1 expression and the apoptotic index (AI). Mcl-1 expression was significantly increased in gastric cancer tissues compared to normal gastric mucosa tissues, and was associated with age, tumor size, stage, depth of invasion, lymph node metastasis and poor survival. Our study showed that Mcl-1 regulates the cell growth and might be a potential prognostic marker for gastric cancer.

Effect and mechanism of miRNA to osteosarcoma cell.

MicroRNA has proved to be low expression in many tumor cells. In addition, it was also proved that as a kind of cancer suppressor gene, miR-199a-3p in miRNA can affect the growth and invasion ability of tumor cells. This paper aims to discuss the effect of miRNA to osteosarcoma cell. It used synthetic mature miR-199a-3p sequence simulants to transfect osteosarcoma cell and took negative contrast sequence (NC mimics) transfection cell as negative contrast. After transfection, qRT-PCR was applied to detect the expression quantity of miR-199a-3p in every group. Western blot method was applied to detect the expression level of MCL-(1) protein and shear situation of PARP in groups of cells. Flow cytometry was used for detecting apoptosis rate of cells and the experimental result was made a statistical analysis. The result shows that in cells from experimental group of transfection miR-199a-3p sequence simulants, expression quantity of mi-R-199a-3p significantly increased while MCL⁻¹ protein expression decreased compared to control group. In addition, shear level of PARP protein and apoptosis rate of cells increased. The differences all had statistical significance (P<0.05). It was concluded that miR-199a-3p can effectively promote the apoptosis rate of osteosarcoma cells.

A measure of DNA sequence similarity by Fourier Transform with applications on hierarchical clustering.

Multiple sequence alignment (MSA) is a prominent method for classification of DNA sequences, yet it is hampered with inherent limitations in computational complexity. Alignment-free methods have been developed over past decade for more efficient comparison and classification of DNA sequences than MSA. However, most alignment-free methods may lose structural and functional information of DNA sequences because they are based on feature extractions. Therefore, they may not fully reflect the actual differences among DNA sequences. Alignment-free methods with information conservation are needed for more accurate comparison and classification of DNA sequences. We propose a new alignment-free similarity measure of DNA sequences using the Discrete Fourier Transform (DFT). In this method, we map DNA sequences into four binary indicator sequences and apply DFT to the indicator sequences to transform them into frequency domain. The Euclidean distance of full DFT power spectra of the DNA sequences is used as similarity distance metric. To compare the DFT power spectra of DNA sequences with different lengths, we propose an even scaling method to extend shorter DFT power spectra to equal the longest length of the sequences compared. After the DFT power spectra are evenly scaled, the DNA sequences are compared in the same DFT frequency space dimensionality. We assess the accuracy of the similarity metric in hierarchical clustering using simulated DNA and virus sequences. The results demonstrate that the DFT based method is an effective and accurate measure of DNA sequence similarity.

Merkel cell carcinoma dependence on bcl-2 family members for survival.

Merkel cell carcinoma (MCC), a rare but aggressive cutaneous neoplasm with high metastatic potential, has a poor prognosis at late stages of disease with no proven chemotherapeutic regimens. Using an enriched culture medium, we established and characterized 11 MCC cell lines for Bcl-2 family profiling and functional studies. Immunoblot analysis revealed collectively high protein levels of prosurvival Bcl-2 members in cell lines and a panel of MCC tumors. Downregulation of individual Bcl-2 proteins by RNAi promoted death in a subset of MCC cell lines, whereas simultaneous inhibition of multiple family members by using the small-molecule antagonist ABT-263 led to a marked induction of cell death in 10 of 11 lines. ABT-263 induced Bax-dependent apoptosis with rapid cleavage of caspase-3 and PARP, regardless of Bcl-2 family profile or the presence of Merkel cell polyomavirus. Furthermore, ABT-263 treatment led to rapid and sustained growth suppression of MCC xenografts from a representative cell line, accompanied by a striking increase in apoptosis. Our results establish that concurrent inhibition of multiple prosurvival Bcl-2 proteins leads to effective induction of apoptosis, and strongly support the concept that targeting MCC dependence on these molecules may be useful therapeutically by reversing an intrinsic resistance to cell death.